G-protein-coupled receptors (GPCRs) play an important role in a multiplicity of physiological processes. They are one of the most important protein families known to date, and it is assumed that in the human genome about 1000 genes code for members of this receptor class. GPCRs have a characteristic structure: they are peptide threads which meander in the form of α-helices seven times through the phospholipid bilayer of the cell membrane, arranging themselves in a circle. It is estimated that about 60% of the pharmaceuticals presently available through prescription bind to GPCRs. This underlines the importance of this receptor class to the pharmaceutical research industry.
G-protein-coupled receptors share a common mechanism of action. Binding of an extracellular ligand leads to a conformational change in the receptor protein that allows it to make contact with a guanine-nucleotide binding protein (G-protein). G-proteins are located on the cytoplasmic side of the plasma membrane and mediate the extracellular signal in the cell interior to trigger various intracellular reactions.
GPCRs are the most important therapeutic target proteins to date. An estimated 40% of the pharmaceuticals prescribed by doctors act as agonists or antagonists of GPCRs. Owing to the size and importance of this protein family and in view of the fact that chemical binding partners for many GPCRs are unknown (orphan GPCRs), it can be assumed that this receptor class will be one of the most important reservoirs for suitable target proteins in the search for novel medicinal substances in the future.
GPCRs are integral membrane proteins that transfer a signal mediated via a mostly hydrophilic signal substance bound to the outer side of the cell into the cell interior via a family of G-proteins. Depending on the receptor specificity and the G-proteins activated thereby, activated GPCRs trigger various signal transduction pathways. Depending on the receptor type, various actions are evoked, all of which lead to the formation of second messengers. Second messengers are intracellular messenger molecules, such as, for example, cAMP, cGMP, and Ca2+, formed in or released into the cytosol in response to an extracellular signal and which trigger reactions in the cell through the activation or deactivation of intracellular proteins. Thus, activation of a membrane-bound adenylate cyclase may lead to an increase in the intracellular cAMP level, and inhibition may lead to a decrease. Stimulation of a cGMP-specific phosphodiesterase may lead to a reduction in the cGMP level. The activated G-protein can also lead, for example, to an increase of Ca2+ or K+ ions by binding to an ion channel. Furthermore, an activated G-protein can affect activation of a phospholipase and thus formation of inositol 1,4,5-trisphosphate and diacylglycerol. This, in turn, leads either to a Ca2+ increase or to activation of a protein kinase C, with further effects in both cases.
The heterotrimeric G-proteins are located on the inside of the plasma membrane. They comprise the three subunits α, β and γ. When an activated receptor makes contact with the G-protein heterotrimer, it dissociates into an α subunit and a βγ complex. Both the activated α subunit and the βγ complex can influence intracellular effector proteins. The G-protein α subunit family is presently divided into four different classes (Gαs, Gαi, Gαq and Gα12 classes).
GPCRs are classified according to the G-proteins that they contact. GPCRs of the Gs class mediate adenylate cyclase stimulation via activation of Gαs and increase the intracellular cAMP concentration. GPCRs of the Gi class mediate adenylate cyclase inhibition via activation of Gαi and decrease intracellular cAMP. GPCRs of the Gq class mediate stimulation of various phospholipase C beta (PLCβ) isoforms via activation of Gαq and lead to hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to give diacylglycerol and inositol 1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from intracellular depots.
Most GPCRs can make contact only with one G-protein α subunit family, and, therefore, are selective for a particular signal transduction pathway. This narrow specificity is a great hindrance to the identification of chemical compounds capable of modulating GPCR-dependent signal transduction pathways.
Moreover, a suitable signal which can be utilized in a screening assay with high sample throughput is obtained only from those signal transduction pathways in which, for example, G-protein activation leads to an increase in the intracellular Ca2+ level.
Hybrid G-proteins with altered receptor specifity and signal transduction pathway linkage may be constructed by joining together parts of various G-proteins using known molecular biology and biochemistry methods.
Hybrid G-proteins are fusion constructs which combine sequences of various Gα subunits within one protein. Thus it is possible, for example by fusion of the Gαi receptor recognition region to the Gαq effector activation region, to prepare a Gαq/i hybrid which receives signals from Gi-coupled receptors but switches on the Gαq-PLCβ signal transduction pathway. Such a hybrid, in which the C-terminal 5 amino acids of Gαq is replaced by the corresponding Gαi sequence (Gαqi5), was first described by Conklin et al., Nature 363, 274-276 (1993).
This “recoupling” of receptors has the advantage that the assay endpoint (increase in intracellular Ca2+ concentration in comparison with adenylate cyclase inhibition) is more readily accessible through measurement methods and can be used in high throughput screening.
However, the disadvantage of the Gαq/Gαi fusion constructs is that they are unable to activate some GPCRs, such as, for example, the SSTR1 receptor qi5 (Conklin et al., Mol. Pharmacol. 50, 885-890 (1996)).
Similarly, fusion constructs between Gαq and Gαs have been described. These too have the disadvantage that they cannot link all Gs-coupled receptors to the PLCβ signal transduction pathway, such as the β2-adrenergic receptor and the dopamine D1 receptor, for example.
Besides C-terminal modifications for altering the linking of receptors to particular signal transduction pathways, an N-terminal modification of Gαq has been described which allows the G-protein to receive and pass on signals from several different receptors. In this Gαq protein, the 6 highly conserved N-terminal amino acids were deleted (Kostenis et al., J. Biol. Chem. 272, 19107-19110 (1997)). This deletion allows the resulting Gq (also called −6q) to receive signals not only from Gq- but also from Gs- or Gi/o-coupled receptors and to pass them on to PLCβ.
This mutant Gα subunit also recognizes receptors such as the SSTR1 somatostatin receptor, the dopamine D1 receptor and the adrenergic β2 receptor. However, even this mutant is unable to recognize the receptor edg5. Moreover, the signal intensity of this mutant is so weak that it is unusable in practice (Kostenis et al., J. Biol. Chem. 272, 19107-19110 (1997)).
Another known Gα subunit is Gα16 which links GPCRs from various functional classes to the PLCβ-Ca2+ signal transduction pathway. Gα16 is a G-protein with broad receptor specificity and has been disclosed in WO 97/48820 (title: Promiscuous G-protein compositions and their use). Gα16 is practically nonselective by nature. But even this subunit is not universally applicable, because receptors such as the edg5 receptor or the SSTR1 somatostatin receptor couple to it only weakly, if at all.
Thus, it would be very useful if a G-protein were available that could be activated by other functional GPCR classes, could also give a sufficiently strong signal in the cell. Such a G-protein could be utilized in a screening assay, such as a high throughput screening assay, to identify compounds modulating GPCRs and/or the appropriate dependent signal transduction pathways, for example a signal such as the increase or decrease in the intracellular Ca2+ concentration.
The object of the present invention is therefore to provide further hybrid G-proteins characterized by having recognizable broad specificity with respect to GPCRs. These G-proteins can be used in screening processes to identify chemical compounds by the coupling of the G-proteins to a signal pathway leading to an increase in the intracellular Ca2+ concentration. In addition, these proteins can be expressed at such a high level that signal intensity is improved.